Pcdiat. Res. 5.-312-328 (1971) cell size growth retardation developmental physiology muscle mass DNA protein malnutrition Review Article Skeletal Muscle Cell Mass and Growth: The Concept of the Deoxyribonucleic Acid Unit DONALD B. CHEEK'1451, ALAN B. HOLT, DONALD E. HILL, AND JAMES L. TALBERT Division of Growth, Children's Medical and Surgical Center, Johns Hopkins Hospital, Baltimore, Maryland, USA Speculation Two important predictions, contradicting current opinion, arise from this review: Removal of the pituitary causes loss of muscle deoxyribonucleic acid (DNA) in the weanling rat. Exercise causes increase in muscle tissue with a commensurate increase in DNA and protein. Therefore, some of the muscle DNA must be in a dynamic state. Calorie restriction (without protein restriction) imposed during the postweanling period of growth in rats does slow cell multiplication but does not cause permanent growth retardation. Therefore, protein restriction, per se, is probably responsible for permanent growth retardation in the experimental animal. Introduction hormones, and exercise. We believe postnatal cyto- plasmic growth of muscle tissue is influenced by pro- Muscle contains a high proportion of the cellular mass tein intake and insulin activity while growth hormone of the body and is of considerable importance to pro- and calorie intake influence the rate of increase in the tein synthesis, especially in larger mammals. It is the number of nuclei. Alterations in the production and purpose of this paper to review past and new informa- tissue response to these hormones can be detected by tion concerning the growth of skeletal muscle and to muscle analysis. Since satellite cells would appear to be support and substantiate the concept of the DNA unit progenitors of myoblasts, factors that influence growth in muscle. This essentially functional concept assumes must influence satellite cell behavior. that each nucleus within the fiber has jurisdiction over a finite mass of cytoplasm. Developmental Information Because the DNA per diploid nucleus is constant, the nuclear popidation of the muscle mass can be ap- The musculature is made up of special cells—muscle praised if the sample analyzed is representative. Data fibers. Muscle fibers arise from myoblasts which origi- will be presented supporting this statement, and meth- nate in the middle germinal layer of the embryo (mes- ods of measuring muscle mass will be discussed. More- enchyme) [10, 100, 103, 128]. over, the ratio of protein to DNA should reflect the Myotubes form by the fusion of multinuclcatcd size or mass of such a functional cell unit. Cell size can cells, and the nuclei within the myotubes are thought then be compared with organ size or, in this instance, not to divide mitotically or amitotically [-1, 123]. Dur- total muscle mass. ing the last trimester of pregnancy muscle nuclei of the Mathematical expressions are presented derived human fetus move to the periphery of the cell (or are from ongoing work concerning increments in the size displaced by the myofibrils). Further postnatal growth and number of such functional muscle units when was assumed to occur by increase in fiber size, or length, they are related, respectively, to chronological age and or both [82]. hotly size during development. Surrounding an entire muscle or smaller muscle The functional DNA unit is influenced by nutrition, bundles or the myofibcr are discrete connective tissue Skeletal muscle cell mass and growth 313 sheaths, and chemical analysis reveals only Wo colln- bers that their nuclei appear, by light microscopy, to gen/g fresh weight [19]. Cells such as histiocytes, fibro- be within the myotube. These workers noted that mi- blasts, ncuronal cells, and adipocytes are present in tosis occurred only in cells outside the myotube and not muscle tissue and contribute by virtue of their nuclei to in any cells that had evidence of myosin synthesis. Ac- the DNA content of each gram of muscle. Work in pro- cording to Lee [81] and to Klinkerfuss [75] the plasma gress by Dr. Charles Friedman in our laboratory indi- membrane surrounding satellite cells can penetrate cates that during growth 25% of the nuclei in human into the sarcoplasm and additional nuclei are added to muscle are outside the myofibcr. The method used for (he myofiber. this determination is that of Dunnill [37]. Satellite cells would appear to be of great impor- Satellite cells according to Mauro [88] are mononu- tance to the understanding of muscle growth and to be cleated cells that are wedged between the basal or responsible for the increments in DNA of muscle dur- plasma membrane of the muscle fiber and the myotu- ing growth. Presumably they are the progenitors of the bule. During embryonic life these cells are abundant; myoblast. however, it is not clear whether they do or do not The ultrastructurc of striated muscle has been re- participate in normal myogencsis [70, 105]. viewed [118] and will not be discussed here except to Reznik [111, 112] found that satellite cells are more point out that arguments relating to the limits of the numerous in areas undergoing regeneration and that extracellular volume in muscle [127, 129, 130] should these cells incorporate tritium-labeled thymidine. He take into account the sarcotubular system [69]. considered them "stem cells" capable of providing my- oblasts for the mature mammal and for regeneration Flistological Approaches to Qu/intitation of Fiber Size of skeletal muscle tissue. and Number lJischofE and Holtzer [5] recently found that my- In 1898 MacCalluin [82] studied the middle third of otubes formed by the union of myoblasts involved cell sartorius muscle in the adult, newborn, and fetus; he recognition and membrane-to-membrane interactions found in the adult a cross-sectional fiber area nine when the cells were in the Gl phase of division. They times that of the newborn. An increase in muscle fiber also recognized that certain surrounding stem cells re- volume occurred after 6 months of gestation, but no mained and suggested that these cells were "prime can- increment in the number of muscle fibers or in the didates for the recruitment of new myoblasts during number of nuclei. In 1902 Godlewski [50] reached a regeneration." They observed that satellite cells could similar conclusion, whereas Schierterdecker [114] in- constitute presumptive myoblasts. sisted that the number of nuclei in muscle fillers in- A review by Kelly and Zachs [74] embraces many creased postnatally. studies showing that secondary and tertiary muscle Tello [125] considered that new fibers probably do cells originate as buds from the walls of primary cells arise in the human postnatally or prenatally. Mont- during fetal life; others found that a tertiary genera- gomery [97] found that in the fetal period in humans tion of muscle cells develops from mononuclear cells the number of fibers (and nuclei) increased, and that in close association with primary generations of my- between week 32 of gestation and 1 months of age, the otubes [2, 32, 31, 93, 98]. Kelly and Zacks [74] empha- number of muscle fibers doubled; between birth and sized the continued presence of undilferentiated cells adulthood, the number of nuclei in the cross-sectional between myotubes that have been described by Mauro areas studied increased by a factor of 10 or more. Con- [88] as being "fibroblast like." It is these undifferen- currently, muscle fiber diameter increases postnatally tiated cells, which lie beneath the basal lamina sur- two- to threefold [9, 36, 59, 137]. rounding the myotubes, that can be classed as belong- In other mammals, up to a 10-fokl increase in fiber ing to the potential replicating population. Several diameter has been shown [29, 51, 60, 63, 73, 121]; incre- investigators recorded that the frequency of satellite ments in sarcomerc units [89, 99, 117] are in the same cells in muscle declines as the fetus reaches term. Such order. cells, according to Wirsen and Larsson [136], are res- The mere measurement of fiber diameter, however, ponsible for the checkerboard pattern of distribution throws little light on the actual increase in fiber mass, of histochemically distinct myofibers when muscle is which is three-dimensional. Inspection of body compo- observed under the microscope. sition changes in the mouse during growth [22] and Shafiq ct al. [116] concluded that undifferentiated consideration of the changes in muscle fiber length and myoblasts and satellite cells are so close to the myofi- sarcomerc number [52] show clearly that sarcomere 314 CHEEK, HOLT, HILL, AND TALBERT number increases in male mice with the adolescent born; at weaning, 26-30%; at sexual maturity, 37- spurt, while muscle fiber length correlates well with the 43%; at 5 months, 41-44%. No sex differences were overall growth of the lean body mass. Thus, measure- demonstrated. Caster ct al. [13], using gravimetric ment of fiber diameter or cross-sectional area yields methods, dissection, and actomyosin determination, very limited information. found a muscle mass of 45% of the body weight in rats The weight of evidence indicates that new fibers can of 320-350 g. arise after birth, and the old thesis that no new nuclei Cheek ct al. [24, 58] determined the muscle mass in appear in muscle postnatally is certainly erroneous. male eviscerated Sprague-Dawley rats without skin or feet (defined as carcass), using three methods which Muscle Mass gave comparable results. They showed that the rela- Muscle mass and body lueight; individual muscle tion between muscle mass and body weight in rats can weight. In cattle, sheep, and pigs [60, 90, 106], the be clearly defined by linear equations (Table I). In weight of a dissected individual muscle, or anatomi- this study muscle mass constituted 80% of fat-free car- cally defined group of muscles, is closely correlated cass, and a similar equation has evolved (Table I) for with the total muscle mass.